High amylose wheat starch and wheat containing the same

ABSTRACT

Wheat starch of the present invention is obtained from endosperm of a seed of wheat which is modified to lack starch granule protein-1 (SGP-1). The wheat starch has an apparent amylose content of about 35% or more. Wheat flour of the present invention is obtained from endosperm of a seed of wheat which is modified to lack SGP-1. Wheat of the present invention is modified to lack SGP-1. The wheat flour and the wheat comprise wheat starch which has an apparent amylose content of about 35% or more.

BACKGROUND OF THE INVENTION

[0001] 1. Field of the Invention:

[0002] The present invention relates to wheat starch having novelproperties and, more particularly, to wheat starch having a highapparent amylose content.

[0003] 2. Description of the Related Art:

[0004] Starch is the major component of the endosperm of a cereal seedsuch as wheat. Wheat starch components can be either amylose oramylopectin. The amylose content of wheat starch is 0% for waxy wheatcultivars and about 22-30% (about 29% on average) for normal (nor-waxy)wheat cultivars.

[0005] Some maize cultivars yield corn starch in which the amylosecontent is as high as about 60-70%. Cornstarch with high amylose contentprovides various industrial applications, such as an adhesive forcardboard, a converging agent for glass fiber and an edible film. aswell as food applications such as ricemeal which is used when makingrice cake.

[0006] Among various rice varieties, Indica rice grain has a higheramylose content than Japonica rice grain. Rice grains having a highamylose content can. be suitably used for pilaf and rice vermicelli.

[0007] Wheat starch with a high amylose content and wheat flourcontaining such wheat starch are expected to provide new industrial andfood applications. Therefore, attempts have been made to produce wheatstarch with increased amylose content using crossbreeding and geneticengineering approaches. However, to the extent the present inventor isaware of, no satisfactory results have been obtained.

[0008] Amylose is an α(1,4)-linked glucose polymer which. is essentiallya linear chain without branching. Amylopectin is a branched glucosepolymer in which branch chains are linked to the main chain of α(1,4)-linked polymer by α(1,6)-linkages. The linear glucose polymers aresynthesized by the action of starch synthases which produces(1,4)-linkages. The (1,6)-linkages of amylopectin are produced by theaction of branching enzymes.

[0009] Studies in pea, maize, and wheat (Denyer et al., Plant J.4:191-198, 1993; Echt and Schwartz, Genetics 99:275-284, 1981; Mu etal., Plant J. 6:151-159, 1994: and Denyer et al., Planta 196:256-265,1995) have shown that some enzymes for starch synthesis are tightlybound to starch granules from seed endosperms of maize and wheat and peaembryo.

[0010] The detailed mechanism for the binding of these enzymes to starchgranules has been unknown. However, it is believed that in wheat, atleast four kinds of proteins, i.e., waxy protein and three starchgranule proteins (SGP-1, SGP-2, SGP-3), are tightly bound to starchgranules and are responsible for starch synthesis. Waxy protein, i.e.,granule-bound starch synthase I (GBSS I) responsible for amylosesynthesis, is the product of the waxy gene (Ainsworth et al., Plant MolBiol. 22:67-82, 1993). SGP-1, -2 and -3 (Yamamori and Endo, Theor Appl.Genet. 93:275-281, 1996) correspond to starch granule-bound isozymes ofabout 100-105 kDa, about 90 kDa and about 77 kDa , respectively,reported by Denyer et al. (Planta, supra). Immunoblotting, amino acidsequencing and detection of starch synthase or branching activities(Denyer et al., Planta, supra: Rahman et al., Aust. J. Plant Physiol.22:793-803, 1995; Takaoka et al., J. Agric. Food Chem. 45:2929-2934,1997) suggest that SGP-2 is a homolog of maize branching enzyme IIb(Fisher et al., Plant Physiol. 102:1045-1046, 1993) and that SGP-3 is ahomolog of maize starch synthase I (Knight et al., Plant J. 14:613-622,1998).

[0011] Immunoblotting studies on about 100-105 kDa protein (SGP-1) didnot detect the protein in the soluble fraction. Thus, SGP-1 isexclusively bound to starch granules (Denyer et al., Planta, supra;Rahman et al., supra) This protein is presumed to be a starch synthasefrom the studies of antiserum recognition, enzymatic activity detectedand homology in amino acid sequences (Denyer et al., Planta, supra;Takaoka et al., supra). However, information regarding the physiologicalfunction of SGP-1 in vivo has been limited. For maize, it has beenreported that an apparent amylose content is increased in a mutant ofdull 1 gene which is presumed to code for starch synthase II (Gao etal., The Plant Cell 10:399-412, 1998). However, there is no substantialamino acid sequence homology between the protein coded by dull 1 (Gao atal., supra) and the protein SGP-1 of wheat (Takaoka et al., supra).Further, the protein coded by dull 1 is present in the soluble fraction.Thus, the starch synthase encoded by dull 1is significantly differentfrom SGP-1.

[0012] A hexaploid wheat has three isozymes of SGP-1, i.e., SGP-A1,SGP-B1 and SGP-D1. The gene coding for SGP-A1, Sgp-A1, is located onchromosome arm 7A, Sgp-B1 on 7B, and Sgp-D1 on 7D (Denyer et al.,Planta, supra). Using SDS-polyacrylamide gel electrophoresis (SDS-PAGE),it has been found that a few wheat cultivars lacked either SGP-A1,-B1 or-D1, but no wheat cultivars lacked two or more SGP-1s (Yamamori andEndo, supra).

SUMMARY OF THE INVENTION

[0013] According to one aspect of this invention, there is providedwheat starch obtained from endosperm of a seed of wheat which ismodified to lack starch granule protein-1 (SGP-1). The wheat starch hasan apparent amylose content of about 35% or more.

[0014] According to another aspect of this invention, there is providedwheat flour obtained from endosperm of a seed of wheat which is modifiedto lack SGP-1. The wheat flour includes wheat starch which has anapparent amylose content of about 35% or more.

[0015] According to still another aspect of this invention, there isprovided wheat which is modified to lack SGP-1. The wheat includes wheatstarch which has an apparent amylose content of about 35% or more.

[0016] In one embodiment, the apparent amylose content may be from about30% to about 45%, preferably from about 37% to about 40%.

[0017] In one embodiment, the wheat may be a hexaploid wheat which lacksSGP-A1, SGP-B1 and SGP-D1 The hexaploid wheat may be obtained bycrossing a first wheat lacking a first protein selected from the groupconsisting of SGP-A1, SGP-B1 and SGP-D1, with a second wheat lacking asecond protein which differs from the first protein and is selected fromthe group consisting of SGP-A1, SGP-B1 and SGP-D1, followed by furthercrossing the cross of the first wheat and the second wheat with a thirdwheat lacking a third protein which differs from the first and secondproteins and is selected from the group consisting of SGP-A1, SGP-B1 andSGP-D1. The hexaploid wheat may be obtained by crossing (i) Chousen 30or Chousen 57, (ii) Turkey 116, and (iii) Kanto 79 in an arbitraryorder.

[0018] Thus, the invention described herein makes possible the advantageof providing wheat starch having a high apparent amylose content.

[0019] This and other advantages of the present invention will becomeapparent to those skilled in the art upon reading and understanding thefollowing detailed description with reference to the accompanyingfigures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0020]FIG. 1 is a photograph of a gel developing an electrophoresispattern of wheat lacking one or more SGP-1s Lane 1 shows Chinese Springas a control; lane 2 shows Turkey 116: lane 3 shows Kanto 79; lane 4shows Chousen 57: and lane 5 shows SGP-1 null wheat.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0021] Hereinafter, the present invention will be described in greaterdetail.

[0022] 1. Definitions

[0023] “SGP-1” is one of several kinds of proteins referred to as starchgranule proteins, or “SGPs”, which are not washed off, but remain boundto, starch granules of wheat seed endosperms during a process forwashing the starch granules with a buffer containing a surfactant,sodium dodecyl sulfate (SDS). Recent reports have shown that SGP-1 is astarch synthase. Hexaploid wheat having genome organization of AABBDDhas three isozymes of SGP-1, i.e., SGP-A1, -B1 and -D1. Tetraploid wheathaving genome organization of AABB has two isozymes of SGP-1, i.e.,SGP-A1 and -B1. These isozymes of SGP-1 can be -detected and identifiedby SDS-gel electrophoreses as three distinguished protein bands.Specifically, SGP-A1, -B1 and -D1 are detected by SDS-gelelectrophoresis as bands of about 115 kDa, about 100 kDa and about 108kDa, respectively (Yamamori and Endo, supra).

[0024] Herein, the terms “SGP-1”, “SGP-A1”, “SGP-B1” and “SGP-D1” areused to denote proteins, while “Sgp-1”, “Sgp-A1”, “Sgp-B1” and “Sgp-D1”are used to denote genes coding for the proteins, SGP-1, SGP-A1, SGP-B1and SGP-D1, respectively.

[0025] The phrase “lacking” SGP-1 as used herein means that any SGP-1protein is not expressed at a level detectable in SDS-gelelectrophoresis. More specifically, it means that the band of theprotein of interest is substantially undetectable by silver stainingwhich is a sensitive method for protein detection.

[0026] The term “apparent amylose content” as used herein refers to anamylose content as measured by calorimetric measurement based on iodinecoloration using an auto-analyzer or by amperometric titration based oniodine affinity. In the context of the present invention, when it isstated that wheat has an apparent amylose content of “about 35% ormore”, for example, means that the wheat has an amylose content of about35% or more as measured under conditions that are substantially the sameas those used in either the colorimetric measurement as described insection (1) of Example 4below or the amperometric titration as describedin section (2) of Example 4 below, or both.

[0027] The wheat starch herein disclosed can also be characterized bymaximum absorbance (λ_(max)) and absorbance at 680 nm (blue value). Asmeasured under substantially the same conditions as those of Example 3below, the wheat starch may have λ_(max) of from about 600 nm to about620 nm, and blue value of from about 0.45 to about 0.55.

[0028] “Wheat” refers to a plant belonging to the genus Triticum. Wheatincludes “hexaploid wheat” which has genome organization of AABBDD,comprised of 42 chromosomes, and “tetraploid wheat” which has genomeorganization of AABB, comprised of 28 chromosomes. Hexaploid wheatincludes T. aestivum, T. spelta, T. macha, T. compactum, T.sphaerococcum, T. vavilovii, and interspecies cross thereof. Tetraploidwheat includes T. durum, T. dicoccoides, T. dicoccum, T. polonicum, andinterspecies cross thereof. A wheat cultivar for use in the presentinvention may belong to any of the above-listed species, and preferablya hexaploid wheat, and more preferably T. aestivum.

[0029] “Modified” wheat as used herein refers to wheat which has beenartificially manipulated to lack SGP-1, and it is intended to excludenaturally-occurring wheat The artificial manipulation of wheat istypically, but not limited to, cross breeding. It may be any otherappropriate manipulation, including mutagenesis and geneticrecombination.

[0030] “Kanto 79/Turkey 116” as used herein refers to a cross obtainedby pollinating Kanto 79 with pollen of Turkey 116. “(Kanto 79/Turkey116)F₂//Chousen 57” as used herein refers to a cross obtained by firstpollinating Kanto 79 with pollen of Turkey 116 to obtain a plant (F₁),self-pollinating the plant (F₁) to obtain a new progeny plant (F₂); andthen pollinating the progeny plant (F₂) with pollen of Chousen 57.

[0031] 2. Production of Wheat Lacking SGP-1 (SGP-1 Null Wheat)

[0032] As a method for completely eliminating all SGP-1 proteins fromhexaploid wheat, the present inventors have developed a novel method aswill be described below It is noted that as a pollen parent for thecrossing process which will be described below, either of the parentwheat cultivars can be used because SGP-1 is coded by a chromosomalgene.

[0033] First, a wheat cultivar lacking only SGP-D1 (Sgp-D1 null), forexample, is crossed with another wheat cultivar lacking only SGP-B1(Sgp-B1 null) so as to obtain F₁ seeds. Since a wheat cultivar isgenerally a homozygote, the obtained F₁ seeds will be heterozygous forboth Sgp-D1 and Sgp-B1, whereby both SGP-D1 and SGP-B1 will be detectedin the F₁ seed endosperms. When F₁ plants which have grown from the F₁seeds are self-pollinated, F₂ seeds will segregate with regard to eachof Sgp-D1 and Sgp-B1 alleles at the probability of one out of four (¼).That is, one out of four F₂ seeds will be null as to the Sgp-B1 gene,and independently, one out of four F₂ seeds will be null as to theSgp-D1 gene. Thus, from the entire F₂ seed population, an F₂ seed beingnull as to both Sgp-D1 and Sgp-B1 is obtained by the probability of oneout of sixteen ({fraction (1/16)}; i.e., ¼ multiplied by ¼),theoretically.

[0034] Starches are purified from distal halves of the obtained F₂grains, and examined for the presence or absence of SGP-D1 and SGP-B1 bysubjecting to electrophoresis so as to select those lacking both SGP-D1and SGP-B1. The. proximal halves corresponding to the selected distalhalves are seeded to obtain plants lacking both SGP-D1 and SGP-B1. Theobtained plant is crossed with another wheat cultivar lacking onlySGP-A1 (Sgp-A1 null) so as to obtain new F₁ seeds. The new F₁ seeds willbe heterozygous for all of Sgp-A1, Sgp-B1 and Sgp-D1, and therefor allof SGP-A1, SGP-B1 and SGP-D1 will be detected in endosperms of the newF₁ seeds. When new F₁ plants which have grown from the new F₁ seeds areself-pollinated, new F₂ seeds will segregate with regard to each ofSgp-A1, Sgp-B1, and Sgp-D1 alleles at the probability of one out of four(¼). Thus, from the entire new F₂ seed population, a new F₂ seed beingnull as to all of Sgp-A1, Sgp-B1 and Sgp-D1 is obtained at theprobability of one out of sixty-four ({fraction (1/64)}; i.e., ¼multiplied by ¼, further multiplied by ¼), theoretically.

[0035] Starches are purified from distal halves of the obtained new F₂grains, and examined for the presence or absence of SGP-A1, -B1 and -D1by subjecting the SGPs to electrophoresis so as to select those lackingall of SGP-A1, -B1 and -D1. The proximal halves corresponding to theselected distal halves are seeded to obtain plants lacking all ofSGP-A1, -B1 and -D1.

[0036] While an exemplary crossing process has been described above, theorder of crossing is not limited to the order described above. Wheatcultivars lacking only SGP-A1 (SGP-A1 null wheat) include Chousen 30,Chousen 57, and the like. Wheat cultivars lacking only SGP-B1 (SGP-B1null wheat) include Kanto 79, and the like. Wheat cultivars lacking onlySGP-D1 (SGP-D1 null wheat) include Turkey 116, and the like. Wheatcultivars lacking SGP-A1, SGP-B1 or SGP-D1 are not limited to thoselisted above. Other such cultivars may be obtained by screeningaccording to the method described in Yamamori and Endo, supra.

[0037] Tetraploid wheat, e.g., durum wheat, which lacks SGP-1 can alsobe obtained in a manner similar to that for SGP-1 null hexaploid wheat.For example, SGP-1 null tetraploid wheat may be produced by firstobtaining hexaploid wheat (2n=42) lacking both SGP-A1 and -B1, crossingthe obtained hexaploid wheat with durum wheat, and then -selectingprogenies being tetraploid (2n=28) and lacking both SGP-A1 and -B1.Alternatively, SGP-1 null tetraploid wheat may be produced by firstcrossing two durum wheat cultivars which lack SGP-A1 and -B1,respectively, self-pollinating the obtained cross, and then selectingprogenies which lack both SGP-A1 and -B1.

[0038] SGP-1 null hexaploid wheat may alternatively be produced bycrossing tetraploid wheat lacking both SGP-A1 and -B1 with Aegilopssquarrosa having genome organization of DD and lacking SGP-D1 so as toobtain triploid individuals, subjecting the obtained triploidindividuals to a doubling of chromosomes such as a colchicine treatmentso as to obtain hexaploid progeny, and then obtaining hexaploidprogenies which lack all of SGP-A1, SGP-B1 and SGP-D1.

[0039] The present invention has been made based on a discovery thatwheat lacking SGP-1 produces novel starch having a high level ofapparent amylose content which, to the extent that the present inventoris aware of, has not been previously known in the art. Wheat starch andwheat flour of the present invention may be obtained by any method withwhich wheat lacking SGP-1 can be produced, and such method is notlimited to the cross breeding as described above. For example, wheatlacking SGP-1 may alternatively be obtained by first treating wheathaving SGP-1 with a mutagen, and then screening the treated wheat plantor progeny of the treated wheat plant obtained by self-pollinating thetreated wheat plant, for the absence of all of SGP-A1, SGP-B1 andSGP-D1. Alternatively, when a wheat plant lacking one or two of SGP-A1,SGP-B1 and SGP-D1 is found in a wheat plant population obtained by emutagenic treatment, crossing process(es) may further be performed usingsuch wheat plant as a parental plant so as to obtain wheat plant lackingSGP-1.

[0040] The mutagen may be any appropriate mutagen including a physicalmutagen such as ionizing radiation and a chemical mutagen. The physicalmutagens include gamma ray, X ray, fast neutron, thermal neutron, betaray, and the like. The chemical mutagens include ethyl methanesulfonate(EMS), N-methyl-N-nitrosourea (MNU), diethyl sulfate (dES), sodium azide(NaN₃), and the like. Appropriate methods for treating wheat plant withsuch mutagens, and appropriate conditions including what kind of wheatmaterial is to be used with such a treatment are known to, and will beselected by, those skilled in the art.

[0041] Moreover, wheat lacking SGP-1 may alternatively be produced byany appropriate genetic engineering approach known in the art, includingprotoplast fusion, homologous recombination, antisense technique, andthe like. Those skilled in the art can appropriately select one of theseand other approaches, and combinations thereof.

[0042] 3. Production of Wheat Starch and Wheat Flour with High AmyloseContent

[0043] Wheat starch having the high amylose content of the presentinvention may be prepared by isolating starch from wheat seed lackingSGP-1 according to any appropriate method known in the art. Wheat flourhaving the high amylose content of the present Invention may be preparedby milling the wheat seed lacking SGP-1 according to any appropriatemethod known in the art.

[0044] Wheat starch and wheat flour of the present invention arebelieved to be novel materials characterized by having a high level ofapparent amylose content which has not been previously known in the art.Such wheat starch and wheat flour may be useful in various industrialand food applications. Moreover, wheat starch of the present inventionmay also be useful for the purpose of researching the correlationbetween the structure of glucose polymer and starch properties.Furthermore, modified wheat of the present invention may be useful as abreeding material for developing wheat which produces starch having anamylose content as high as that of maize (60%-70%).

EXAMPLES Example 1 Production of Wheat Lacking SGP-1 (SGP-1 Null Wheat)

[0045] 1. Plant Material

[0046] To produce a wheat which lacks SGP-1 (SGP-1 null wheat), thefollowing four parental wheat (Triticum aestivum L.) cultivars wereused: Chousen 30 (C 30) and 57 (C 57) lacking SGP-A1; Kanto 79 (K 79)lacking SGP-B1 and Turkey 116 (T 116) lacking SGP-D1 (see Table 1Y.

[0047] First, T 116 and K 79 were crossed to obtain F₁ seeds. F₁ plantswhich grew from the F₁ seeds were self-pollinated to obtain F₂ seeds.Starches were purified from the distal half of the F₂ seeds.SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed usingthe purified starches so as to examine the presence or absence of SGP-D1and -B1. As a result, F₂ seeds lacking both SGP-D1 and -B1 from cross K79/T 116 were selected. Purification of the starches and SDS-PAGE willbe described in greater detail below.

[0048] F₂ plants which grew from the selected F₂ seeds lacking bothSGP-D1 and -B1 were pollinated by either of C 30 and C 57, both lackingSGP-A1, to obtain new F₁ seeds. New F₁ plants grown from the new F₁seeds were self-pollinated to obtain new F₂ seeds. Starches werepurified from the distal half of the new F₂ seeds. SDS-polyacrylamidegel electrophoresis (SDS-PAGE) was performed using the purified starchesso as to examine the presence or the absence of SGP-A1, -B1 and -D1. Asa result, from the cross (K 79/T 116)F₂//C 30 or C 57, variant progeny(new F₂ plant) lacking SGP-1 was selected. TABLE 1 Alleles for Sgo-1 inwheat materials used to produce wheat with no SGP-1 (SGP-1 null wheat)Sgp-1 Wheat −A1 −B1 −D1 SGP-1 null b b b Turkey 116 a a b Kanto 79 a b aChousen 57 b a a Chouscn 30 b a a Chinese Spring a a a Norin 61 a a a

[0049] In Sgp-1 alleles, a indicates standard allele in cv-ChineseSpring which produces the protein coded by the gene, while b indicatesnull allele which does not produce the coding protein. The allele -A1belongs to A genome, -B1 to B genome and to -D1 to D genome. Four wheats(Turkey 116- Chousen 30) were used to produce SGP-1 null wheat. Twocultivars (Chinese Spring and Norin 61) were controls for analyzingstarch properties.

[0050] 2. Starch and Starch Granule Preparation

[0051] Starches from the distal half of F₂ seeds for screening wereprepared according to Sulaiman and Morrison (J. Cereal Sci. 12:53-61(1990)), using 80% CsCl.

[0052] For characterization of starch, starch granules were preparedaccording to Echt and Schwartz, supra. Hammer-crushed wheat seeds werehomogenized in a protein extraction buffer (55 mM Tris/HCl, pH 6.8, 2.3%SDS, 5% 2-mercaptoethanol and 10% glycerol). This suspension was passedthrough a 50 μm nylon mesh to remove large seed coats. Aftercentrifugation at 13,500 rpm for 2 min, a yellowish layer on whitestarch pellet was removed by spatula, and the remaining white starchpellet was suspended in the extraction buffer. This procedure wasrepeated twice, then the starch was washed twice by distilled water andtwice by acetone and air dried.

[0053] 3. SDS-polyacrylamide Gel Electrophoresis

[0054] SDS-polyacrylamide gel electrophoresis of starch granule proteins(SGPs) was performed as described by Yamamori and Endo, supra. An amountof 5 mg of starch prepared from ten (10) mature grains or 5 mg of starchfrom a distal half of F₂ grain was gelatinized in 70 μl of the proteinextraction buffer by heating for 5 min. After centrifugation for 5 minat 13,500 rpm, the supernatant (15 μl) was subjected to electrophoresis.For the resolution gel, acrylamide in a concentration of 12.5% andBIS-acrylamide in a low concentration (acrylamide/BIS-acrylamideconcentration of 30.0.135) were used. Proteins were visualized by silverstaining (Silver stain kit; Wako Pure Chemical Industries, Ltd. Japan).

[0055] For characterization of starch, cultivar Chinese Spring or Norin61 having all of SGP-A1, -B1 and -D1 were used as controls.

[0056] 4. Results

[0057] SDS-PAGE analysis of 968 new F₂ seeds from the cross (Kanto79/Turkey 116)F₂//Chousen 30 or Chousen 57 found that four seeds yieldedno SGP-1. New F₂ seeds were classified into eight categories based onSgp-1 alleles or the presence or absence of SGP-A1, -B1 and -D1. Sincethe three genes, Sgp-A1, -B1 and -D1 are located on differentchromosomes, the expected ratio for the eight categories is27:9:9:9:3:3:3:1 (see Table 2). However, the observed number did not fitthe expected ratio (X²=14.26, P<0.05). Seed fertility of F₃ plantsderived from new F₂ plants (SGP-1 null) was 94%, while fertility ofcultivar Chinese Spring was 97%. This shows fertility of the SGP-1 nullwheat was normal. The SGP-1 null wheat used in Examples 2-4 below wasobtained from (Kanto 79/Turkey 116)F₂//Chousen 57. TABLE 2 Segregationof F₂ from (Kanto 79,Trukey 116)F₂// Chousen 30 or Chousen 57 Alleles ofSgp-1 Number of F₂ Expected X² -A1 -B1 -D1 seeds observed ratio value aa a 424 27  0.60 b a a 150 9  1.41 a b a 125 9  0.91 a a b 143 9  0.35 ab b 47 3  0.06 b a b 38 3  1.20 b b a 37 3  1.55 b b b 4 1  8.18 Total968 64 14.26*

Example 2 Decrease in Other Starch Granule-Bound Proteins in SGP-1 NullWheat

[0058] In addition to SGP-1, wheat starch granules carry threegranule-bound proteins, i.e., waxy protein, SGP-2 and SGP-3. In new F₂,F₃ and F₄ seeds of the SGP-1 null wheat, SGP-2 and -3 decreasedconsiderably while the waxy protein did not, as observed on a gel ofSDS-PAGE. The result for the F₄ seeds is seen in lane 5 of FIG. 1. Toexamine how much the SGP-2 and -3 decreased in the SGP-1 null wheat, 1,½, ¼, ⅛, {fraction (1/16)} and {fraction (1/32)} sample volumes of thecultivar Chinese Spring were subjected to electrophoresis and thethickness of SGP-2 and -3 bands detected by silver staining werecompared to one volume from the SGP-1 null wheat. As a result, it wasfound that the elimination of SGP-1 was accompanied with a decrease ofboth SGP-2 and -3 to about {fraction (1/16)} as compared with thecultivar Chinese Spring.

Example 3 Measurement of Blue Value and λ_(max) of Wheat Lacking SGP-1(SGP-1 Null Wheat)

[0059] To characterize starch components, the present inventor measuredthe blue value (absorbance at 680 nm) and maximum absorbance (λ_(max))of iodine-starch complex from the SGP-1 null wheat (F₄ seeds), itsparents and cultivar Chinese Spring (see Table 3). Higher blue valueindicates that the apparent amylose content of the SGP-1 null wheat washigher than those of the others.

[0060] The absorbance at 680 nm (blue value) and maximum. absorbance(λ_(max)) of the iodine-starch complex were determined according toKonishi et al. (Agric Biol. Chem. 49:1965-1971, 1985). An amount of 10mg of starch was gelatinized in 1 ml of 1N NaOH for one hour at 40° C.,and neutralized by 9 ml of {fraction (1/9)} M acetic acid. Then, 1 mg ofgelatinized and neutralized starch was mixed with 2 mg of I₂ and 20 mgKI, and distilled water was added to make a 25 ml solution. Absorptioncurves of starch-iodine complexes were measured at 500-700 nm, and bluevalue and λ_(max) were recorded.

Example 4 Measurement of Amylose Content of SGP-1 Null Wheat

[0061] To confirm that wheat lacking SGP-1 has a high apparent amylosecontent, the amylose content was measured by calorimetric method andamperometric titration as follows.

[0062] (1) Colorimetric measurement based on Iodine coloration wasperformed following the method of Kuroda et al. (Jpn. J. Breed. 39(Suppl. 2):142-143, 1989) using an auto-analyzer (Bran Lubbe. Co.). Anamount of 35 mg of starch was gelatinized in 5 ml of 0.75 N NaOH and 25%aqueous ethanol, and neutralized by acetic acid. Absorbance at 600 nm ofstarch iodine complex was measured by colorimeter. For control, twowheat starches were used. A first control, wheat starch purchased fromWako Pure Chemicals Ltd. (Japan) contained 31.2% amylose as determinedby the auto-analyzer using potato amylose and amylopectin as standards,and a second control, waxy wheat starch contained 0.6% amylose.

[0063] The amylose content of the starch from the SGP-1 null wheat wasas high as 37.3% (see Table 3). In contrast, Norin 61 and Chinese Springhad an amylose content of 28.2% and 29.6%, respectively. Thus, theamylose content of the SGP-1 null wheat starch was higher than those ofcultivars Norin 61 and Chinese Spring by about 8% to 9%. The three wheatcultivars used as crossing parents, i.e., Turkey 116, Kanto 79 andChousen 57, had amylose contents ranging from 23.9% to 30.3%.

[0064] (2) Amperometric titration (Fukuba and Kainuma, “Quantificationof amylose and amylopectin” in Starch Science Handbook (Nakamura M. andSuzuki S., eds) Tokyo: Asakura Shoten, pp 174-179, 1977) was performedusing defatted starch with an iodine amperometric titration device(Model 3-05, Mitamura Riken Kogyo, Japan). Amylose content of the starchwas calculated by assuming that 20 mg of iodine can bind to 100 mg ofpure wheat amylose. The starch concentration of the solution used wasdetermined by the phenol-sulfuric acid method (Dubois et al., Anal.Chem. 28:350-356, 1956) with glucose as a standard.

[0065] The amylose content of the starch from the SGP-1 null wheat was37.3% (see Table 3). In contrast,-Norin 61 and Chinese Spring had anamylose content of 26.6% and 29.3%, respectively. Thus, the amylosecontent of the SGP-1 null starch was higher than that of cultivars withSGP-1, Norin 61 and Chinese Spring by about 8% to 11%. The three wheatcultivars used as crossing parents had amylose contents ranging from23.54 to 29.8%. TABLE 3 Maximum absorbance (λmax), absorbance at 680 nm(blue value) of starch-iodine complex and amylose content of wheats (F₄seeds) λmax Blue Amylose content (%) Wheat (nm) value ColorimetricTitration SGP-1 null (F₄) 602 ± 6 0.485 ± 0.023 37.3 ± 0.8 37.3 ± 0.8Turkey 116 589 ± 5 0.370 ± 0.011 30.3 ± 0.2 29.8 ± 0.5 Kaoto 79 565 ± 00.307 ± 0.004 23.9 ± 0.5 23.5 ± 0.1 Chousen 57 591 ± 1 0.365 ± 0.00329.4 ± 0.1 28.1 ± 0.2 Chinese Spring 586 ± 3 0.358 ± 0.013 29.6 ± 0.129.3 ± 0.3 Norin 61 —¹⁾ — 28.2 ± 0.1 26.6 ± 0.2

[0066] Values are means±SD from three replicates for two controls andthree parental wheats. For SGP-1 null, values from eight (titration),ten (calorimetric) and 15 (λ_(max) and blue value) replicates wereindicated.

[0067] The above-mentioned measurement results all indicate that theapparent amylose content of the SGP-1 null wheat starch is considerablyhigher than that of normal wheat starch.

[0068] Various other modifications will be apparent to and can bereadily made by those skilled in the art without departing from thescope and spirit of this invention. Accordingly, it is not intended thatthe scope of the claims appended hereto be limited to the description asset forth herein, but rather that the claims be broadly construed.

What is claimed is:
 1. Wheat starch obtained from endosperm of a seed ofwheat which is modified to lack starch granule protein-1 (SGP-1),wherein the wheat starch has an apparent amylose content of about 35% ormore.
 2. The wheat starch of claim 1, wherein the apparent amylosecontent is from about 37% to about 40%.
 3. The wheat starch of claim 1,wherein the wheat is a hexaploid wheat which lacks SGP-A1, SGP-B1 andSGP-D1.
 4. The wheat starch of claim 3, wherein the hexaploid wheat isobtained by crossing a first wheat lacking a first protein selected fromthe group consisting of SGP-A1, SGP-B1 and SGP-D1, with a second wheatlacking a second protein which differs from the first protein and isselected from the group consisting of SGP-A1, SGP-B1 and SGP-D1,followed by further crossing the cross of the first wheat and the secondwheat with a third wheat lacking a third protein which differs from thefirst and second proteins and is selected from the group consisting ofSGP-A1, SGP-B1 and SGP-D1.
 5. The wheat starch of claim 3, wherein thehexaploid wheat is obtained by crossing (i) Chousen 30 or Chousen 57,(ii) Turkey 116, and (iii) Kanto 79 in an arbitrary order.
 6. Wheatflour obtained from endosperm of a seed of wheat which is modified tolack SGP-1, comprising wheat starch which has an apparent amylosecontent of about 35% or more.
 7. The wheat flour of claim 6, wherein theapparent amylose content is from about 37% to about 40%.
 8. The wheatflour of claim 6, wherein the wheat is a hexaploid wheat which lacksSGP-A1, SGP-B1 and SGP-D1.
 9. The wheat flour of claim 8, wherein thehexaploid wheat is obtained by crossing a first wheat lacking a firstprotein selected from the group consisting of SGP-A1, SGP-B1 and SGP-D1,with a second wheat lacking a second protein which differs from thefirst protein and is selected from the group consisting of SGP-A1,SGP-B1 and SGP-D1, followed by further crossing the cross of the firstwheat and the second wheat with a third wheat lacking a third proteinwhich differs from the first and second proteins and is selected fromthe group consisting of SGP-A1, SGP-B1 and SGP-D1.
 10. The wheat flourof claim 8, wherein the hexaploid wheat is obtained by crossing (i)Chousen 30 or Chousen 57, (ii)Turkey 116, and (iii) Kanto 79 in anarbitrary order.
 11. Wheat which is modified to lack SGP-1, comprisingwheat. starch which has an apparent amylose content of about 35% ormore.
 12. The wheat of claim 11, wherein the-apparent amylose content isfrom about 37% to about 40%.
 13. The wheat of claim 11, which is ahexaploid wheat which lacks SGP-A1, SGP-B1 and SGP-D1.
 14. The wheat ofclaim 13, wherein the hexaploid wheat is obtained by crossing a firstwheat lacking a first protein selected from the group consisting ofSGP-A1, SGP-B1 and SGP-D1, with a second wheat lacking a second proteinwhich differs from the first protein and is selected from the groupconsisting of SGP-A1, SGP-B7 and SGP-D1, followed by further crossingthe cross of the first wheat and the second wheat with a third wheatlacking a third protein which differs. from the first and secondproteins and is selected from the group consisting of SGP-A1, SGP-B1 andSGP-D1.
 15. The wheat of claim 13, wherein the hexaploid wheat isobtained by crossing (i) Chousen 30 or Chousen 57, (ii) Turkey 116, and(iii) Kanto 79 in an arbitrary order.